Efficient targeting of specific cells of interest to achieve stable, durable transduction of these cells is a basic goal of gene therapy strategies. To this end, retroviral vectors have been widely utilized for a number of applications in the laboratory. Inserting ligands or single chain antibodies into the retroviral receptor-binding envelope subunit has been the most common approach to alter and/or restrict the host range of retroviral vectors. Bridging vector and target cell by antibodies or ligands is another approach. These modified envelope proteins appear to have specific binding properties but low fusion activity resulting in inefficient entry into target cells. This may be due to the fact that with retroviral infection, viral binding and fusion are structurally linked events, which must occur in an ordered fashion to be successful. Therefore, many of these strategies have suffered from inconsistent specificity and low viral titers as a result of these modifications of the retroviral envelope. Recent studies in the Chen laboratory have resulted in a new approach to developing an efficient retroviral vector with specific targeting properties. Lentiviral-based, recombinant Sindbis virus pseudovirions displaying the IgG-binding domain of protein A (Z domain) in the E2 region of the viral envelope have been developed. One of these, HRCMVEGFP (ZZ Sindbis) has been shown to enhance targeting and transduction of HLA class I and CD4+ cells up to 30-fold using specific monoclonal antibodies to these cell surface markers compared to no antibody. Optimization of these vectors using site-directed mutagenesis studies to alter the Sindbis virus envelope E2 domains known to be responsible for host cell binding is the focus of this research project. These studies will seek among other things to reduce the nonspecific infection (background) associated with these vectors. Insertion of the IgG-binding domain of protein G as well as avidin into the Sindbis virus envelope E2 region will also be carried out to extend the utility of this vector. The host range and cell specificity characteristics of these vectors will also be explored by examining differential targeting of CD4 and HLA class I on a variety of cell types. Finally, based on the results of these initial studies, a model for targeting the viral-encoded marker, thy 1.2, HIV-1-infected cells will serve as proof-of-principle of the capabilities of this vector system. Future studies using HIV-1/Sindbis virus vectors to target HIV-1-infected cells as a potential gene therapy application of this vector system is proposed. I am seeking a KO8 grant to allow me to expand my HIV clinical research career with basic science training to enable me to undertake translational studies directed at HIV therapeutics.